CN107796895B - Construction method and application of on-line extraction high performance liquid characteristic spectrum of fresh cordyceps sinensis - Google Patents
Construction method and application of on-line extraction high performance liquid characteristic spectrum of fresh cordyceps sinensis Download PDFInfo
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Abstract
The invention belongs to the technical field of chromatographic analysis, and particularly discloses a construction method for on-line extraction of a high performance liquid characteristic spectrum from fresh cordyceps sinensis, which is characterized by comprising the following steps of: (1) preparing an extractor; (2) extracting on line; (3) detecting by high performance liquid chromatography tandem mass spectrometry; (4) and (5) constructing a characteristic map. The on-line extraction-high performance liquid chromatography system provided by the invention has the advantages of simple device, simple and convenient operation, less required sample amount and less time consumption, greatly promotes the application of the method in actual production, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of chromatographic analysis, in particular to a construction method of a high performance liquid characteristic spectrum extracted from fresh cordyceps sinensis on line and application of the high performance liquid characteristic spectrum in the on-line extraction and analysis of effective components of traditional Chinese medicinal materials.
Background
Cordyceps sinensis is a dry complex of stroma and larval cadaver that is parasitic on insect larvae of the family Hepialidae, the fungus Cordyceps sinensis (BerK.) Sacc.) of the family Clavicipitaceae. Has sweet and neutral nature and flavor, can enter lung and kidney channels, has the effects of tonifying kidney and lung, stopping bleeding and reducing phlegm, and is used for treating kidney deficiency and essence deficiency, impotence and spermatorrhea, soreness and pain of waist and knees, chronic cough and dyspnea due to deficiency, overstrain cough and hemoptysis, and the like. Modern researches show that the cordyceps sinensis contains active substances such as nucleoside, protein, polysaccharide, sterol and the like, and has the activities of resisting tumors, regulating immunity, resisting inflammation, resisting aging and the like.
The cordyceps sinensis is a rare traditional Chinese medicine, and in recent years, due to the fact that market demand is gradually increased and resources are very deficient, price is high, and adulterants, confusing products and counterfeit products are increased in the market. Since the 2000 edition of Chinese pharmacopoeia, adenosine was collected as a content determination index of Cordyceps quality standard. Research shows that besides adenosine, uridine, inosine, guanosine, adenine, guanine and other nucleoside components are also index components of cordyceps sinensis, and compared with content measurement with a single index, the characteristic fingerprint spectrum of the nucleoside components of cordyceps sinensis can comprehensively reflect the quality of medicinal materials.
The prior literature reports that the sample preparation method for analyzing the cordyceps sinensis nucleoside components mostly adopts conventional off-line extraction methods such as a thermal reflux extraction method, a Soxhlet extraction method, an immersion extraction method, an ultrasonic extraction method and the like, and the methods not only have large medicinal material and solvent consumption, but also have long extraction time and more operation steps. Therefore, the establishment of a rapid and convenient sample preparation method is beneficial to the improvement of the content determination technology of the cordyceps sinensis nucleoside components. At present, relevant research on a pretreatment system of an online liquid chromatography medium exists, but certain limitation exists, for example, a device provided by a liquid medium pretreatment device for online chromatographic analysis (patent number ZL 200720074371.X) can realize pretreatment of the liquid medium and also can achieve the purposes of large flow, blockage prevention and basic bubble elimination, but the device is not really connected with a liquid chromatograph to form an online analysis system, and cannot really realize online analysis; in addition, there are also reports related to liquid chromatography online desalting, enrichment and mass spectrometry online combined system (patent No. ZL 200610134981.4) and a method for analyzing trace organic matters in water by solid phase extraction-liquid chromatography online combined analysis (patent No. ZL 200610023536.0). However, these studies have corresponding limitations and incompleteness. In recent years, literature reports show that the online sample extraction-high performance liquid analysis technology has the advantages of high speed, sample saving, high automation degree and the like compared with the traditional offline extraction technology, and has been successfully applied to component analysis of traditional Chinese medicinal materials, but the reported literature methods generally need to add six-way valves, pressurizing devices, enrichment columns and other devices in a conventional liquid phase system, so that certain difficulties exist in practical production and application. For example, patent No. ZL 201310107475.6 provides an online analysis method for high performance liquid chromatography, which includes a sample pretreatment system, a high performance liquid chromatograph, an editable logic controller, and a touch screen, and realizes online detection of sample content and component ratio, but the system has many devices and is complicated to operate.
Disclosure of Invention
In order to make up the defects of the prior art and meet the existing requirements, the invention aims to provide the establishing method for the online extraction of the high-performance liquid characteristic spectrum of the fresh cordyceps sinensis. The invention is realized by the following technical scheme:
on one hand, the method for establishing the on-line extraction high performance liquid characteristic spectrum of the fresh cordyceps sinensis comprises the following steps:
(1) preparation of an extractor:
preparation of mixed control extractors: respectively taking a proper amount of reference substances, precisely weighing, adding a solvent for dissolving, and preparing a mixed reference substance solution; precisely measuring a mixed reference substance solution, adding the mixed reference substance solution into a pre-column core filled with diatomite, drying, covering two ends of the column core with microporous filter membranes, and installing the column core in matched pre-column sleeves to obtain an extractor of the mixed reference substance;
preparing a test sample extractor: taking a proper amount of a test sample, adding diatomite, and fully grinding; accurately weighing the uniformly mixed sample and diatomite, filling the sample and diatomite into an empty pre-column core, covering two ends of the sample and diatomite by using microporous filter membranes, and installing the sample and diatomite into a matched pre-column sleeve to obtain an extractor of the sample;
(2) online extraction: the extractor is connected to the front of a protective column in a high performance liquid chromatograph and is placed in a column incubator; conveying the extraction solvent through a liquid phase high pressure pump, and performing online extraction in an extractor to obtain an extracting solution; the extraction process is triggered by the water absorption of an automatic sample injector; the column oven is provided with a first temperature control module and a second temperature control module; the first temperature control module controls the temperature of the extractor, and the second temperature control module controls the temperature of the protective column and the chromatographic column;
(3) high performance liquid chromatography tandem mass spectrometry detection: enabling the extracting solution obtained in the step (2) to enter a protective column, and then carrying out high performance liquid chromatography tandem mass spectrometry detection through a chromatographic column; the high performance liquid chromatography conditions are as follows: the flow rate is 1-2.5 mL/min; mobile phase a was water and mobile phase B was 0.1% acetic acid-methanol solution; the gradient elution was: 0-3.5 min: 0% of B; 3.5-15.5 min: 0% B → 30% B; 15.5-25 min: 30% of B;
(4) constructing a characteristic spectrum: determining chromatographic peaks in the online detection result of the sample extractor according to the online detection result of the mixed reference substance extractor, and establishing a characteristic spectrum by using the following 8 chromatographic peaks:
peak 1 is uracil, retention time is 2.76min, mass spectrometry data is 113[ M + H ]]+m/z；
Peak 2 is uridine, retention time 3.84min, mass spectra data 245[ M + H]+m/z；
Peak 3 was inosine, retention time was 8.66min, and mass spectral data was 269[ M + H]+m/z；
Peak 4 was guanine, retention time 9.24min, mass spectral data 152[ M + H]+m/z；
Peak 5 is guanosine, retention time 9.75min, and mass spectral data 284[ M + H ]]+m/z；
Peak 6 is adenine, retention time is 13.51min, mass spectra data is 136[ M + H]+m/z；
Peak 7 is adenosine, retention time 15.30min, mass spectrometry data 268[ M + H]+m/z；
Peak 8 was 2' -deoxyadenosine, retention time was 17.55min, mass spectral data was 252[ M + H ]]+m/z。
Preferably, the preparation method of the mixed reference substance extractor comprises the following steps: respectively taking appropriate amount of reference substances uracil, guanine, adenine, uridine, inosine, guanosine, adenosine and 2' -deoxyadenosine, precisely weighing, adding water according to the weight-volume ratio of 1: 10-50 mg/mL, and dissolving to prepare a mixed reference substance solution with the concentration of 20-100 mug/mL; precisely measuring the mixed reference substance solution, adding into the pre-column core filled with diatomite, drying, covering the two ends of the column core with microporous filter membranes, and installing in a matched pre-column sleeve to obtain the extractor filled with the mixed reference substance.
Preferably, the sample is fresh cordyceps sinensis, and the amount of the sample and the diatomite which are uniformly mixed is precisely weighed to be 5-7 mg.
Preferably, the extraction solvent of the on-line extraction is water.
Preferably, the extraction time of the online extraction is 2.5-4 min.
Preferably, the extraction time of the on-line extraction is 3.5 min.
Preferably, the flow rate in step (3) is 1.5 mL/min.
Preferably, the first temperature control module controls the temperature of the extractor to be 70 ℃; the extraction solvent is water, and the online extraction time is 3.5 min; the extracting solution is controlled by a second temperature control module to be cooled, enters a protective column, and is subjected to high performance liquid chromatography tandem mass spectrometry detection through a chromatographic column; the extraction process was triggered by the autosampler sucking 10 μ L of water.
Preferably, the guard column used in the online extraction is: agilent Zorbax SB-AQ (4.6 mm. times.12.5 mm, 5 μm), the chromatographic column is Agilent Zorbax SB-AQ (4.6 mm. times.150 mm, 5 μm), and the temperature of the second temperature control module is controlled to 20 ℃.
Preferably, the detection wavelength of the high performance liquid chromatography tandem mass spectrometry is 260 nm; the detector is a diode array detector; the mass spectrum conditions are as follows: quadrupole mass spectrometry with a split ratio of 2: 1; a positive ion mode; selective ion detection (SIM) m/z: 112 (Cytosine, [ M + H ]]+) 113 (uracil, [ M + H ]]+) 127 (thymine, [ M + H ]]+) 136 (adenine, [ M + H ]]+) 137 (hypoxanthine, [ M + H ]]+) 152 (guanine, [ M + H ]]+) 243 (thymidine, [ M + H ]]+) 244 (Cytidine, [ M + H ]]+)、245[M+H]+(uridine, [ M + H ]]+) 268 (adenosine, [ M + H ]]+) 269 (inosine, [ M + H)]+) 284 (guanosine, [ M + H ]]+) 229 (deoxyuridine, [ M + H ]]+) 252 (2' -deoxyadenosine, [ M + H ]]+) And 268 (deoxyguanosine, [ M + H ]]+) (ii) a Electrospray ion source: the flow rate of the drying gas (nitrogen) is 10L/min, the temperature of the drying gas is 350 ℃, and the pressure of the atomizing gas is 35 psi.
Preferably, the method for constructing the high performance liquid characteristic spectrum of the fresh cordyceps sinensis on-line extraction can be applied to on-line extraction analysis of the effective components of the traditional Chinese medicinal materials.
The invention has the beneficial effects that:
(1) the on-line extraction-high performance liquid chromatography system provided by the invention can complete on-line analysis and detection of the sample by only connecting one extractor with the high performance liquid chromatograph, has the advantages of simple device, simple and convenient operation, small required sample amount and short time consumption, greatly promotes the application of the method in actual production, and has good application prospect.
(2) The method adopts the online extraction of the components in the fresh cordyceps sinensis for the first time, and can finish the analysis and detection of the sample within 25min to obtain 8 main chromatographic peaks, and the method has good peak shape and good separation effect, and can provide reference for the quality control of the fresh cordyceps sinensis.
Drawings
FIG. 1 is a schematic diagram of an on-line extraction high performance liquid system.
FIG. 2 is a high performance liquid chromatogram of the mixed control.
FIG. 3 is a high performance liquid chromatography chromatogram of fresh Cordyceps sinensis.
FIG. 4 is a high performance liquid chromatography chromatogram of dried Cordyceps sinensis.
FIG. 5 is a high performance liquid chromatography chromatogram of Cordyceps militaris.
Note: 1 is uracil; 2 is uridine; 3 is inosine; 4 is guanine; 5 is guanosine; 6 is adenine; 7 is adenosine; 8 is 2' -deoxyadenosine;
9 is unknown compound 1; 10 is deoxyuridine; 11 is unknown compound 2; 12 is unknown compound 3; 13 is unknown compound 4; 14 is unknown compound 5, 15 is unknown compound 6; 16 is unknown compound 7; 17 is unknown compound 8.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
For further understanding of the features and technical means of the present invention, as well as the specific objects and functions attained by the present invention, the advantages and spirit of the present invention will be further understood by reference to the following detailed description and the accompanying drawings. It should be understood that the following examples are only illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it should be understood that various modifications and changes may be made by those skilled in the art after reading the present disclosure, and such equivalents may fall within the scope of the invention as defined by the appended claims.
Example 1: on-line extraction of fresh cordyceps sinensis and establishment of high performance liquid characteristic spectrum
1.1 instruments and reagents
The instrument comprises the following steps: high performance liquid chromatograph: agilent model 1260, containing an online degasser, a quaternary pump, an autosampler, a column oven, a DAD detector; quadrupole mass spectrometer: agilent model 6130; one-tenth-of-ten-thousandth analytical balance: mettler Toledo XS 204; parts per million analytical balance: mettler Toledo XP 26; an online extractor: phenomenex, Security Guard; protection of the column: agilent Zorbax SB-AQ, specification 4.6mm × 12.5, 5 μm; a chromatographic column: agilent Zorbax SB-AQ, specification 4.6mm × 150, 5 μm.
Reagent: methanol: spectrum corporation, chromatographically pure; acetic acid: atlas company, chromatographically pure; ultrapure water: preparing a Milli-Q ultrapure water system; comparison products: uracil, guanine, adenine, uridine and inosine are purchased from China food and drug testing research institute; adenosine was purchased from Wuhan Yuancheng Biotechnology Ltd; guanosine was purchased from Beijing technology, Inc.; 2' -deoxyadenosine was purchased from carbofuran technologies, Inc.
Fresh cordyceps sinensis: from Guangdong Dongyuang pharmaceutical Co.
1.2 analytical methods
(1) Preparation of mixed control extractors: taking a proper amount of uracil, guanine, adenine, uridine, inosine, guanosine, adenosine and 2' -deoxyadenosine, precisely weighing, adding water according to the weight-volume ratio of 1: 10-50 mg/mL, and dissolving to prepare a mixed reference solution with the concentration of 20-100 mu g/mL; precisely measuring the mixed reference substance solution, filling the mixed reference substance solution into a pre-column core containing diatomite, drying, covering two ends of the column core with microporous filter membranes, and installing the column core in matched pre-column sleeves to obtain the extractor filled with the mixed reference substance.
(2) Preparing a test sample extractor: adding a proper amount of ground fresh cordyceps sinensis into diatomite according to the weight ratio of 1:2 of the fresh cordyceps sinensis to the diatomite, and fully grinding; precisely weighing 5-7 mg of a mixture of fresh cordyceps sinensis and diatomite, filling the mixture into an empty pre-column core, covering two ends of the empty pre-column core with microporous filter membranes, and installing the empty pre-column core in a matched pre-column sleeve to obtain an extractor filled with a test sample.
(3) Online extraction: connecting the extractor with sample to high performance liquid chromatograph (shown in FIG. 1), and placing in 70 deg.C column oven; a protective column and a chromatographic column are connected with the outlet of the on-line extractor; the extraction solvent is water, and is delivered by high performance liquid high pressure pump, and is heated by column oven, and then is subjected to on-line extraction by extractor for 3.5 min; cooling the extract in a column incubator on the other side, introducing into a protective column, and performing high performance liquid chromatography tandem mass spectrometry detection on the extract by a chromatographic column; the extraction process is triggered by 10 mu L of water absorbed by an automatic sample injector;
(4) high performance liquid chromatography tandem mass spectrometry detection: chromatographic conditions are as follows: the chromatographic column is Agilent Zorbax SB-AQ (AQ)4.6mm × 150mm, 5 μm); the flow rate is 1.5 mL/min; the column temperature is 20 ℃; mobile phase a was water and mobile phase B was 0.1% acetic acid-methanol solution; the gradient elution procedure was: 0-3.5 min: 0% of B; 3.5-15.5 min: 0% B → 30% B; 15.5-25 min: 30% of B; the detection wavelength is 260 nm; the detector is a diode array detector. Mass spectrum conditions: quadrupole mass spectrometry with a split ratio of 2: 1; a positive ion mode; selective ion detection (SIM) M/z 112 (cytosine, [ M + H ]]+) 113 (uracil, [ M + H ]]+) 127 (thymine, [ M + H ]]+) 136 (adenine, [ M + H ]]+) 137 (hypoxanthine, [ M + H ]]+) 152 (guanine, [ M + H ]]+) 243 (thymidine, [ M + H ]]+) 244 (Cytidine, [ M + H ]]+)、245[M+H]+(uridine, [ M + H ]]+) 268 (adenosine, [ M + H ]]+) 269 (inosine, [ M + H)]+) 284 (guanosine, [ M + H ]]+) 229 (deoxyuridine, [ M + H ]]+) 252 (2' -deoxyadenosine, [ M + H ]]+) And 268 (deoxyguanosine, [ M + H ]]+) (ii) a Electrospray ion source: the flow rate of the dry gas (nitrogen) is 10L/min, the temperature of the dry gas is 350 ℃, and the pressure of the atomization gas is 35 psi;
(5) and (3) detection results: analyzing according to the above chromatographic conditions to obtain high performance liquid chromatogram of the mixed reference substance and fresh Cordyceps, as shown in FIG. 2 and FIG. 3. Through comparing the high performance liquid chromatograms of the mixed reference substance and the fresh cordyceps sinensis and confirming the ion fragments extracted from the nucleoside substances reported in the literature, 12 main chromatographic peak signals are obtained, 8 components are identified as the nucleoside substances, the other 4 components are identified as unknown compounds, and the retention time and mass spectrum data of each chromatographic peak are shown in tables 1 and 2.
TABLE 1 control chromatographic peak retention time and Mass Spectrometry data
TABLE 2 retention time and mass spectrum data of characteristic chromatographic peak of fresh Cordyceps
Note: "-" indicates no signal was detected and Rib indicates ribose.
Example 2: on-line extraction high performance liquid chromatography tandem mass spectrometry analysis of chemical components in dry cordyceps sinensis and cordyceps militaris
(1) Preparing a test sample extractor: the same as in the first embodiment.
(2) Online extraction: the same as in the first embodiment.
(3) High performance liquid chromatography tandem mass spectrometry detection: the same as in the first embodiment.
(4) And (3) detection results: analyzing according to the above chromatographic conditions to obtain high performance liquid chromatogram of dried Cordyceps and Cordyceps militaris, as shown in FIG. 3 and FIG. 4. Comparing the high performance liquid chromatograms of the mixed reference substance, the dry cordyceps sinensis and the cordyceps militaris, and confirming the ion fragments extracted from the nucleoside substances according to literature reports, wherein 12 main chromatographic peak signals are obtained in the high performance liquid chromatogram of the dry cordyceps sinensis, and 7 of the main chromatographic peak signals are the nucleoside substances; the high performance liquid chromatogram of Cordyceps militaris totally obtains 8 main chromatographic peaks, 5 of which are nucleoside substances, and retention time and mass spectrum data of each chromatographic peak are shown in tables 3 and 4.
TABLE 3 identification result of fingerprint characteristic peak of dried Cordyceps sinensis
Note: "-" indicates no signal was detected and Rib indicates ribose.
TABLE 4 fingerprint characteristic peak identification result of Cordyceps militaris
Note: "-" indicates no signal was detected and Rib indicates ribose.
Claims (4)
1. A construction method for on-line extraction of a high performance liquid characteristic spectrum of fresh cordyceps sinensis is characterized by comprising the following steps:
(1) preparation of an extractor:
preparation of mixed control extractors: respectively taking a proper amount of reference substances, precisely weighing, adding a solvent for dissolving, and preparing a mixed reference substance solution; precisely measuring a mixed reference substance solution, adding the mixed reference substance solution into a pre-column core filled with diatomite, drying, covering two ends of the column core with microporous filter membranes, and installing the column core in matched pre-column sleeves to obtain an extractor of the mixed reference substance;
preparing a test sample extractor: taking a proper amount of a test sample, adding diatomite, and fully grinding; accurately weighing the uniformly mixed sample and diatomite, filling the sample and diatomite into an empty pre-column core, covering two ends of the sample and diatomite by using microporous filter membranes, and installing the sample and diatomite into a matched pre-column sleeve to obtain an extractor of the sample;
(2) online extraction: the extractor is connected to the front of a protective column in a high performance liquid chromatograph and is placed in a column incubator; conveying the extraction solvent through a liquid phase high pressure pump, and performing online extraction in an extractor to obtain an extracting solution; the extraction process is triggered by the water absorption of an automatic sample injector; the column oven is provided with a first temperature control module and a second temperature control module; the first temperature control module controls the temperature of the extractor, and the second temperature control module controls the temperature of the protective column and the chromatographic column;
(3) high performance liquid chromatography tandem mass spectrometry detection: enabling the extracting solution obtained in the step (2) to enter a protective column, and then carrying out high performance liquid chromatography tandem mass spectrometry detection through a chromatographic column; the high performance liquid chromatography conditions are as follows: the flow rate is 1-2.5 mL/min; mobile phase a was water and mobile phase B was 0.1% acetic acid-methanol solution; the gradient elution was: 0-3.5 min: 0% of B; 3.5-15.5 min: 0% B → 30% B; 15.5-25 min: 30% of B;
(4) constructing a characteristic spectrum: determining chromatographic peaks in the online detection result of the sample extractor according to the online detection result of the mixed reference substance extractor, and establishing a characteristic spectrum by using the following 8 chromatographic peaks:
peak 1 is uracil, retention time is 2.76min, mass spectrometry data is 113[ M + H ]]+m/z；
Peak 2 is uridine, retention time 3.84min, mass spectra data 245[ M + H]+m/z；
Peak 3 was inosine, retention time was 8.66min, and mass spectral data was 269[ M + H]+m/z；
Peak 4 was guanine, retention time 9.24min, mass spectral data 152[ M + H]+m/z；
Peak 5 is guanosine, retention time 9.75min, and mass spectral data 284[ M + H ]]+m/z；
Peak 6 is adenine, retention time is 13.51min, mass spectra data is 136[ M + H]+m/z；
Peak 7 is adenosine, retention time 15.30min, mass spectrometry data 268[ M + H]+m/z；
Peak 8 was 2' -deoxyadenosine, retention time was 17.55min, mass spectral data was 252[ M + H ]]+m/z；
Wherein the test sample is fresh cordyceps sinensis, and the amount of the test sample and the diatomite which are uniformly mixed is precisely weighed to be 5-7 mg;
wherein the first temperature control module controls the temperature of the extractor to be 70 ℃; the extraction solvent is water, and the online extraction time is 3.5 min; the extracting solution is controlled by a second temperature control module to be cooled, enters a protective column, and is subjected to high performance liquid chromatography tandem mass spectrometry detection through a chromatographic column; the extraction process is triggered by 10 mu L of water absorbed by an automatic sample injector;
wherein the protection column used is: agilent Zorbax SB-AQ, 4.6mm × 12.5mm, 5 μm, chromatographic column Agilent Zorbax SB-AQ, 4.6mm × 150mm, 5 μm, second temperature control module temperature control 20 ℃.
2. The method of claim 1, wherein the mixed control extractor is prepared by: respectively taking appropriate amount of reference substances uracil, guanine, adenine, uridine, inosine, guanosine, adenosine and 2' -deoxyadenosine, precisely weighing, adding water according to the weight-volume ratio of 1: 10-50 mg/mL, and dissolving to prepare a mixed reference substance solution with the concentration of 20-100 mug/mL; precisely measuring the mixed reference substance solution, adding into the pre-column core filled with diatomite, drying, covering the two ends of the column core with microporous filter membranes, and installing in a matched pre-column sleeve to obtain the extractor filled with the mixed reference substance.
3. The method of claim 1, wherein the flow rate in step (3) is 1.5 mL/min.
4. The method of claim 1, wherein the detection wavelength of high performance liquid chromatography tandem mass spectrometry is 260 nm; the detector is a diode array detector; the mass spectrum conditions are as follows: quadrupole mass spectrometry with a split ratio of 2: 1; a positive ion mode; selective ion detection, m/z: 112/cytosine/[ M + H ]]+113/uracil/[ M + H ]]+127/thymine/[ M + H ]]+136/adenine/[ M + H ]]+137/hypoxanthine/[ M + H ]]+152/guanine/[ M + H ]]+243/thymidine/[ M + H [ ]]+244/cytidine/[ M + H ]]+、245[M+H]+Uridine/[ M + H ]]+268/adenosine/[ M + H ]]+269/inosine/[ M + H ]]+284/guanosine/[ M + H ]]+229/deoxyuridine/[ M + H ]]+252/2' -deoxyadenosine/[ M + H ]]+And 268/deoxyguanosine/[ M + H ]]+(ii) a Electrospray ion source: drying gas: nitrogen at a flow rate of 10L/min, a drying gas temperature of 350 ℃ and an atomizing gas pressure of 35 psi.
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